Pluripotent stem cells have the potential to produce differentiated cell types comprising all somatic tissues and organs. Treatment of diabetes using cell therapy is facilitated by the production of large numbers of cells that are able to function similarly to human islets. Accordingly, there is need for producing these cells derived from pluripotent stem cells, as well as reliable methods for purifying such cells.
Proteins and other cell surface markers found on pluripotent stem cell and cell populations derived from pluripotent stem cells are useful in preparing reagents for the separation and isolation of these populations. Cell surface markers are also useful in the further characterization of these cells.
In one example, WO2009131568 discloses a method of purifying a gut endoderm cell comprising: a) exposing a population of cells derived from pluripotent stem cells comprising a gut endoderm cell to a ligand which binds to a cell surface marker expressed on the gut endoderm cell, wherein said cell surface marker is selected from the group consisting of CD49e, CD99, CD165, and CD334; and b) separating the gut endoderm cell from cells derived from pluripotent stem cells which do not bind to the ligand, thereby purifying said gut endoderm cell.
In another example, WO2010000415 discloses the use of an antibody that binds to the antigen TNAP, or functional fragments of the antibody, alone or in combination with an antibody that binds to CD56, or functional fragments of the antibody, for the isolation of stem cells having adipocytic, chondrocytic and pancreatic differentiation potential.
In another example, U.S. Pat. No. 7,371,576 discloses the discovery of a selective cell surface marker that permits the selection of a unique subset of pancreatic stems cells having a high propensity to differentiate into insulin producing cells or into insulin producing cell aggregates.
In another example, U.S. Pat. No. 7,585,672 discloses a method to enrich a culture derived from human embryonic stem cells for cells of endoderm and pancreatic lineages, the method comprising the steps of (a) culturing intact colonies of human embryonic stem cells to form whole, intact embryoid bodies surrounded by visceral yolk sac (VYS) cells, wherein the human embryonic stem cells express Oct-4, surface stage-specific embryonic antigen-3/4 (SSEA 3/4) and epithelial cell adhesion molecule (EpCAM); (b) culturing the embryoid bodies of step (a) under conditions that permit the embryoid body cells to differentiate into a cell population containing cells of the endoderm and pancreatic lineages; (c) dispersing the cell population of step (b) into single cells; (d) selecting against the expression of SSEA 3/4 positive cells to remove undifferentiated cells from the cells of step (c); (e) selecting against the expression of SSEA-1 positive cells to remove VYS cells from the remaining cells of step (d); and (f) selecting from among the remaining cells of step (e) for the expression of EpCAM positive cells to enrich for cells of endoderm and pancreatic lineages.
U.S. Pat. No. 7,585,672 also discloses a method to enrich a culture derived from human embryonic stem cells for cells of endoderm and pancreatic lineages, the method comprising the steps of (a) culturing intact colonies of human embryonic stem cells to form whole, intact embryoid bodies surrounded by visceral yolk sac (VYS) cells, wherein the human embryonic stem cells express Oct-4, surface stage-specific embryonic antigen-3/4 (SSEA 3/4) and epithelial cell adhesion molecule (EpCAM); (b) culturing the embryoid bodies of step (a) under conditions that permit the embryoid body cells to differentiate into a cell population containing cells of the endoderm and pancreatic lineages; (c) treating the cell population of step (b) with an effective amount of fibroblast growth factor 10 (FGFI 0); and (d) dispersing the cell population of step (c) into single cells enriched for cells of endoderm and pancreatic lineages (e) selecting against the expression of SSEA-3/4 positive cells to remove undifferentiated stem cells from the cells of step (d); (f) selecting against the expression of SSEA-1 positive cells to remove VYS cells from the cells of step (e); and (g) selecting from among the remaining cells of step (f) for the expression of EpCAM positive cells to enrich for cells of endoderm and pancreatic lineages.
U.S. Pat. No. 7,585,672 also discloses an enrichment method for the creation of a stem cell derived cell population which does not have tumorigenic capability comprising the steps of (a) culturing intact colonies of human embryonic stem cells to form whole, intact embryoid bodies surrounded by visceral yolk sac (VYS) cells, wherein the human embryonic stem cells express Oct-4, surface stage-specific embryonic antigen-3/4 (SSEA 3/4) and epithelial cell adhesion molecule (EpCAM); (b) culturing the embryoid bodies of step (a) under conditions that permit the embryoid body cells to differentiate into a cell population containing cells of the endoderm and pancreatic lineages; (c) dispersing the cell population of step (b) into single cells; (d) selecting against the expression of SSEA 3/4 positive cells to remove undifferentiated cells from the cells of step (c); (e) selecting against the expression of SSEA-1 positive cells to remove VYS cells from the cells of step (d); and (f) selecting from among the remaining cells of step (e) for the expression of EpCAM positive cells, the resulting cells not forming teratomas when injected in immunocompromised mice.
In another example, US20050260749 discloses a method to enrich a culture derived from stem cells for cells of endoderm and pancreatic lineages, the method comprising the steps of culturing stem cells into the formation of embryoid bodies; and selecting among embryoid bodies for the expression of the species appropriate cell surface stage-specific embryonic and culturing only the embryoid bodies which do not express cell surface stage-specific antigen for differentiation into endoderm and pancreatic cells.
In another example, US20100003749 discloses an isolated pancreatic stem cell population, wherein the pancreatic stem cell population is enriched for CD133+CD49f+ pancreatic stem cells.
US20100003749 further discloses the isolation of pancreatic stem cells from primary pancreatic tissue occurs by selecting from a population of pancreatic cells, pancreatic-derived cells, or gastrointestinal-derived cells for cells that are CD133+, CD49f+, or CD133+CD49f+; removing the cells that are CD15+, wherein the remaining cells are CD15−; introducing the remaining cells to a serum-free culture medium containing one or more growth factors; and proliferating the remaining cells in the culture medium.
In another example, Dorrell et at state: “We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or α-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of α, β, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.” (Stem Cell Research, Volume 1, Issue 3, September 2008, Pages 155-156).
In another example, Sugiyama et al state: “We eventually identified two antigens, called CD133 and CD49f, useful for purifying NGN3+ cells from mice. CD133 (also called prominin-1) is a transmembrane protein of unknown function and a known marker of haematopoietic progenitor and neural stem cells. CD49f is also called α6-integrin, and a receptor subunit for laminin. By combining antibodies that recognize CD133 and CD49f, we fractionated four distinct pancreatic cell populations. Immunostaining and RT-PCR revealed that the CD49fhigh CD133+ cell population (‘fraction I’, 50% of input) comprised mainly differentiated exocrine cells that express CarbA. The CD49flow CD133− fraction (‘fraction III’, 10% of input) included hormone+ cells expressing endocrine products like insulin and glucagon. By contrast, the CD49flow CD133+ fraction (called ‘fraction II’, 13% of input) contained NGN3+ cells, but not hormone+ cells. Approximately 8% of fraction II cells produced immunostainable NGN3. In the CD49f−CD133− fraction (‘fraction IV’, 25% of input), we did not detect cells expressing NGN3, CarbA or islet hormones.” (Diabetes, Obesity and Metabolism, Volume 10, Issue s4, Pages 179-185).
In another example, Fujikawa et al state: “When CD45−TER119− side-scatterlow GFPhigh cells were sorted, α-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-α6 and -β1, but negative for c-Kit and Thy1.1.” (Journal of Hepatlogy, Vol 39, pages 162-170).
In another example, Zhao et al state: “In this study, we first identified N-cadherin as a surface marker of hepatic endoderm cells for purification from hES cell-derivates, and generated hepatic progenitor cells from purified hepatic endoderm cells by co-culture with murine embryonic stromal feeders (STO) cells. These hepatic progenitor cells could expand and be passaged for more than 100 days. Interestingly, they co-expressed the early hepatic marker AFP and biliary lineage marker KRT7, suggesting that they are a common ancestor of both hepatocytes and cholangiocytes. Moreover, these progenitor cells could be expanded extensively while still maintaining the bipotential of differentiation into hepatocyte-like cells and cholangiocyte-like cells, as verified by both gene expression and functional assays. Therefore, this work offers a new in vitro model for studying liver development, as well as a new source for cell therapy based on hepatic progenitors.” (PLoS ONE 4(7): e6468. doi:10.1371/journal.pone.0006468).
In another example, Cai et at state: “To further increase the PDX1+ cell purity, we sorted the activin A-induced cells using CXCR4 . . . , a marker for ES cell-derived endodermal cells. Sorting with CXCR4 enriched the endodermal cell population because nearly all the cells in the CXCR4+ population were positive for the endodermal cell marker SOX17, and >90% of the cells were positive for FOXA2.” (Journal of Molecular Cell Biology Advance Access originally published online on Nov. 12, 2009. Journal of Molecular Cell Biology 2010 2(1):50-60; doi:10.1093/jmcb/mjp037).
In another example, Koblas et at state: “We found that population of human CD133− positive pancreatic cells contains endocrine progenitors expressing neurogenin-3 and cells expressing human telomerase, ABCG2, Oct-3/4, Nanog, and Rex-1, markers of pluripotent stem cells. These cells were able to differentiate into insulin-producing cells in vitro and secreted C-peptide in a glucose-dependent manner. Based on our results, we suppose that the CD133 molecule represents another cell surface marker suitable for identification and isolation of pancreatic endocrine progenitors”. (Transplant Proc. 2008 March; 40(2):415-8)
In another example, Sugiyama et al state: “we found CD133 was expressed by NGN3+ cells. CD133 appeared to be localized to the apical membrane of pancreatic ductal epithelial cells.” (PNAS 2007 104:175-180; published online before print Dec. 26, 2006, doi:10.1073/pnas.0609490104).
In another example, Kobayashi et al state: “The embryonic pancreatic epithelium, and later the ductal epithelium, is known to give rise to the endocrine and exocrine cells of the developing pancreas, but no specific surface marker for these cells has been identified. Here, we utilized Dolichos Biflorus Agglutinin (DBA) as a specific marker of these epithelial cells in developing mouse pancreas. From the results of an immunofluorescence study using fluorescein-DBA and pancreatic specific cell markers, we found that DBA detects specifically epithelial, but neither differentiating endocrine cells nor acinar cells. We further applied this marker in an immunomagnetic separation system (Dynabead system) to purify these putative multi-potential cells from a mixed developing pancreatic cell population. This procedure could be applied to study differentiation and cell lineage selections in the developing pancreas, and also may be applicable to selecting pancreatic precursor cells for potential cellular engineering.” (Biochemical and Biophysical Research Communications, Volume 293, Issue 2, 3 May 2002, Pages 691-697).
Identification of markers expressed by cells derived from pluripotent stem cells would expand the understanding of these cells, aid in their identification in vivo and in vitro, and would enable their positive enrichment in vitro for study and use. Thus, there remains a need for tools that are useful in isolating and characterizing cells derived from pluripotent stem cells, in particular, cells expressing markers characteristic of the pancreatic endocrine lineage.